Anti‐inflammatory effect of mesenchymal stem cells on hepatocellular carcinoma in the xenograft mice model

Abstract Background Hepatocellular carcinoma (HCC) is the fifth most diagnosed cancer and the second leading cause of cancer‐related deaths worldwide. Sorafenib is the standard treatment used in the advanced stages of HCC. Cell therapy with mesenchymal stem cells (MSCs)‐based cell therapy has proven effective in immune regulation and tumour growth inhibition. Objectives In this study, we investigated the anti‐inflammatory effect of MSCs on HCC xenografts. Methods Human HepG2 cell lines were subcutaneously implanted into the flank of 12 nude mice, divided into three groups: the control group, the IV group (intravenous MSCs injection) and the local group (local MSCs injection). Mice were sacrificed 6 weeks after tumour implantation, and tumours were resected entirety. Quantitative real‐time polymerase chain reaction (qRT‐PCR) measured the gene expression of inflammatory markers, including tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐1α and IL‐10. Aspartate transaminase (AST), alanine transaminase (ALT) and urea levels were measured using spectrophotometry to ensure the safety of MSC therapy. Results Gene expressions for all three inflammatory markers were reduced in both MSCs groups compared to the control group. AST, ALT and urea levels remained in normal ranges. Conclusions MSC therapy can reduce inflammation in HCC xenograft mouse models.


INTRODUCTION
Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide (Hajighasemlou et al., 2020) which commonly develops following a prolonged chronic hepatitis infection (Berasain et al., 2009;Coussens & Werb, 2002). The progressive increase in HCC incidence and mortality is mainly due to viral hepatitis B and C. Despite recent advances in diagnostic tools, most HCCs are still first diagnosed in advanced stages where curative treatments are no longer an option (Lencioni et al., 2010).
Human multipotent mesenchymal stem cells (MSCs) have been isolated from various adult tissues, such as bone marrow, skeletal muscle, synovium, dental pulp, liver, brain, placenta, umbilical cord and adipose tissue (Campagnoli et al., 2001;Kim et al., 2010). Lacking immunogenicity, robust immunosuppressive and anti-inflammatory properties, promoting angiogenesis and reducing apoptosis are fascinating features that have prompted their wide application in regenerative medicine (Boomsma & Geenen, 2012;Garcia-Castro et al., 2008;Sanchez et al., 2011). In addition, MSCs can produce a high level of indoleamine 2,3dioxygenase (IDO1), a tryptophan catabolising enzyme that mediates immune tolerance by limiting the availability of the essential amino acid tryptophan and generating toxic metabolites for T cells (Meisel et al., 2004 (Dominici et al., 2006).
This study aimed to investigate the anti-inflammatory effect of MSCs on HCC xenografts in athymic nude mice.

Xenograft models
A total of 12 male athymic nude mice (nu/nu; C57BL/6), aged 6-8 weeks, were purchased from Omid Institute for Advanced Biomodels (Tehran, Iran). The mice were housed and maintained in an individually ventilated cage system under optimised hygienic conditions. The average temperature of each cage was 23 • C with a relative humidity of 65%. Animals had free access to autoclaved commercial diet and water.

Analysis of biochemical factors
Blood samples were collected and centrifuged at 800 relative centrifugal force (RCF) to separate the plasma. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and urea were determined using an automated biochemical analyser (Mindray) to assess the safety of the intervention.

Primer design for quantitative real-time polymerase chain reaction (qRT-PCR)
All gene sequences were obtained from the National Center for Biotechnology Information (NCBI) database, and primers were designed using Primer-BLAST online software (NCBI).

Statistical analysis
One-way analysis of variance was applied to check the distribution of biomarkers, and after that, Dennett's test was performed in case of significant results. SPSS software was used to analyse the data, and the 2 -ΔΔCT method was used to calculate the relative transcript level. qRT-PCR results were analysed by the Rotor-Gene Q software using the Kruskal-Wallis H test.

Analysis of biochemical factors
Serum AST, ALT and urea levels were in their normal ranges, with no statistically significant difference between groups (Table 2).

qRT-PCR
The qRT-PCR results showed inhibition of IL-1α, IL-10 and TNF-α expression in both intravenously and locally MSCs-treated groups. The relative quantification of gene expression for IL-1α was 0.09 for the IV TA B L E 2 Plasma levels of biochemical markers of the liver and kidney function to assess the safety of cell therapy and 0.24 for the locally administered MSC groups (p < 0.05; Figure 2).
For IL-10, these values equalled 0.07 and 0.014 for IV and locally administered MSC, respectively (p < 0.01; Figure 3). As for TNF-α, the relative gene expression equalled 0.03 and 0.04 for IV and local groups, respectively (p < 0.001; Figure 4). The quantification for each marker was compared relative to controls.

Tumour size
The size of the tumours were also measured every 2-3 days after implantation. No significant difference was found among the F I G U R E 2 Comparison of TNF gene expression among groups. A statistically significant reduction was found in TNF expression in both systemic and locally administered groups as compared to control (***p < 0.001). MSC, mesenchymal stem cell; TNF, tumour necrosis factor; CTRL, control; IV, intravenously (systemically administered MSC); LOC, locally administered MSC (in tumour margin)

F I G U R E 3
Comparison of IL-1α gene expression among groups. A statistically significant reduction was found in IL-1 expression in both systemic and locally administered groups when compared to control (*p < 0.05). IL-1, interleukin-1; MSC, mesenchymal stem cell; CTRL, control; IV, intravenously (systemically administered MSC); LOC, locally administered MSC (in tumour margin) average tumour sizes in different groups. Figure 5 depicts the changes in tumour size across time in all three groups.

DISCUSSION
One of the ways to investigate the anti-inflammatory effect of MSCs on HCC xenografts is by determining the expression of various inflammatory mediators such as IL-1α, IL-2, IL-4, IL-8, IL-10, TNF-α and transforming growth factor-β (TGF-β) . In this study, we chose to investigate the expression of IL-1α, IL-10 and TNF-α, due to their key role in immune regulation, inflammatory responses, tissue remodelling, cell motility, cell cycle and apoptosis (Malik &Kanneganti, 2018 andKitaura et al., 2022). Expert opinions are controversial on the role of MSCs in inflammation and tumour progression (Voronov et al., 2014). However, it can be better considered by investigating the interplay between MSCs and the inflammatory milieu (Murray, 2006).
We have previously studied the homing of systematically and locally administered hPMSCs into HCC xenograft models and demonstrated the safety of hPMSCs. We also report the superior privilege of the locally administered cells to migrate to the tumour tissues (Hajighasemlou et al., 2018).
The commonly used medications for inflammation control include non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids, which inhibit multiple inflammation enzymes and mediators. Anti-inflammatory medications are also used in cancer treatment to control the symptoms and act as adjuvants for cytotoxic medications.
One mechanism by which MSCs can inhibit tumour growth is through the different mediators secreted by these cells, which is supported by a study on animal models of human hepatoma, where an increase in tumour cell apoptosis was observed through the downregulation of Bcl-2, c-Myc and proliferating cell nuclear antigen (PCNA) (Qiao et al., 2008).
In a study on mouse liver fibrosis models, a single intravenous administration of stem cells derived from human exfoliated deciduous teeth (SHEDs) removed the fibrotic scar. The researchers suggested that SHEDs serve this effect by suppressing the expression of pro-inflammatory mediators (TNF-α, IL-1β and iNOS) and also by inducing apoptosis in hepatic stellate cells while selectively protecting parenchymal hepatocytes (Hirata et al., 2016).
Another cytokine secreted by MSCs is IL-6 which acts as both proinflammatory and anti-inflammatory cytokines (Scherzad et al., 2015) studied the effect of MSCs on two head and neck cell lines (FaDu and HLaC78) via their interactions with cytokines; they found an inhibition in malignant cell proliferation when IL-6 was added to the culture (Jiang et al., 2011). However, the inflammatory process in malignancies is relatively nonspecific, where we can see an increased expression of cytokines like IL-4, IL-5 and especially IL-1α and TNF-α, whereas the levels of IL-2 are usually decreased (Baier et al., 2005). Taraxacum officinale (TO), commonly known as dandelion, has frequently been used to remedy women's diseases and livers' and gallbladders' disorders. Its anti-tumour properties have also been elucidated in a study on the human hepatoma cell line, HepG2, where it induced apoptosis by increasing the levels of IL-1α and TNF-α in those cells (Koo et al., 2004).
Investigating the effect of microRNA-22 on HCC, researchers detected an increased expression of IL-1α in tumour-adjacent tissues in the male subset, inversely correlated with the expression of oestrogen receptor α. Researchers further revealed that IL-1α was higher in the early stages of tumourigenesis but decreased along with its development (Jiang et al., 2011).

CONCLUSION
Tumour progression can be adversely affected by unregulated inflammation; this property of tumour cells can benefit from controlling the milieu of inflammation using MSCs.

ACKNOWLEDGEMENTS
We would like to thank all our technical assistants for their commitment to this study.

CONFLICT OF INTEREST
Authors have no conflict of interest to declare.

FUNDING
Tehran University of Medical Science and Cancer Research Center of Cancer Institute of Iran (Shams cancer charity) provided financial support.

ETHICAL APPROVAL
Applied treatments in this study were approved by the Ethical Committee of TUMS.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.